The strength and specificity of the avidin-biotin interaction have led to its widespread use in a variety of bioanalytical applications. The avidin-biotin complex has been used in affinity chromatography, affinity cytochemistry, cell cytometry, blotting, diagnostics, drug delivery, etc.
The biotin-avidin interaction is attractive because avidin is a small stable protein that binds four biotin molecules, and biotin is somewhat hydrophilic and easily conjugated to other molecules giving products that complex with avidin. Given the attractiveness of the biotin-avidin interaction, researchers have developed a significant number of assays utilizing biotin and avidin. These methods include microbiological, calorimetric, enzymatic, radiometric, electrochemical, and fluorescent approaches.
European Patent Application Publication No. 0 451 810 published Oct. 16, 1991 describes hapten-biotin conjugates useful in a competitive homogeneous immunoassay in which agglutination occurring during the reaction is evaluated by turbidimetric or nephelometric measurements.
U.S. Pat. No. 4,228,237, issued to Hevey et al. on Oct. 14, 1980, describes a method for determining the presence of a ligand in a liquid medium utilizing enzyme-labeled avidin and a biotinylated reagent wherein the ligand to be detected is contacted with an insoluble phase containing specific binding substance for the ligand.
Green, Biochem. J., 94: 23c-24c (1965) and Green, Methods in Enzymology, (McCormick and Wright, Eds.), Vol. 18, Part A, pages 418-424 (1970) describe the quantification of avidin by measuring changes in the optical absorbance of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) upon binding to avidin; biotin can be quantitated by measuring reversal of these changes upon addition of biotin. Disadvantages of this method include relatively low sensitivity and potential interference from a variety of naturally occurring chromophores.
Mock et al., Analytical Biochemistry, 151: 178-181 (1985) describe a fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid which results in concomitant quenching of the fluorescence.
Schray et al., Anal. Chem., 60: 853-855 (1988), describe a fluorescence polarization assay utilizing a biotin-fluorescein conjugate. Polarization varies as a function of avidin concentration and, at fixed avidin levels, as a function of competing biotin concentrations. This system is quite complicated requiring multiple reagent co-optimization.